Approach 1: Understand and improve on the protective efficacy of ALVAC & gp120 vaccination. In the last several years we have investigated vaccine approaches for HIV, based on DNA alone or in combination with poxvirus-based vaccines, and found that these vaccine modalities are able to elicit mainly memory T-cells, and when combined with gp120 protein boost can induce high titer binding antibodies. Vaccination induced anti-envelope antibodies in all vaccinees and CD4+ and CD8+ T-cell responses. Approximately, one third of the vaccinated macaques were protected from SIVmac251 acquisition, whereas the remaining infected vaccinees progressed to disease. The magnitude of vaccine induced SIVmac251 specific T-cell responses and binding antibodies were not significantly different in protected or infected animals. However, sera from protected animals had higher avidity antibodies to gp120, recognized the variable envelope region V2, and reduced SIVmac251 infectivity in cells that express high level of alpha4 beta7 integrins, suggesting a functional role of antibodies to V2. Mucosal exposure of vaccinated macaques to SIVmac251 faithfully reproduced results in humans, suggesting that this model is instrumental in the development of more efficacious vaccines for HIV. Approach 2: We investigated the impact of SIVmac251 challenge dose on the efficacy of a DNA prime/ALVAC-SIV/ gp120 protein boost vaccine regimen. Infection was neither prevented nor modified following a single high dose of challenge of the immunized macaques. However, exposure to a tenfold lower dose resulted in protection from SIVmac251 acquisition and modulated pathogenesis (decrease CD4+ T-cell loss) in animals that did become infected. The challenge dose did not affect expression of inflammatory genes in the gut during acute infection, but at viremia set point, a significant down regulation of interferon responsive genes and up regulation of genes involved in B and T-cell responses was observed only in vaccinated animals exposed to a lower dose of SIVmac251. In these animals we also found a significant correlation between vaccine induced T-cell responses and protection from CD4+ T-cell loss. We conclude that challenge virus dose can influence relative vaccine efficacy in the Non Human Primate (NHP) model Approach 3: We compared the relative efficacy against simian AIDS of three vaccine regimens that elicited similar frequency of SIV specific CD4+, CD8+ but differed in the level of antibody responses to the gp120 envelope protein. While two of the vaccine regimens protected from high levels of SIV replication only during the acute phase of infection, the third strategy protected during both the acute and the chronic phase of infection and was associated with a better reconstitution of CD4+ T-cells at mucosal sites. All three vaccine regimens elicited equivalent frequencies of SIV-specific T-cells but differed in the elicitation of antibodies to the SIV gp120. The vaccinated group with durable protection had the highest titer of binding antibody to gp120, persistent Gag CD8+ CM9+ effector memory with low expression of surface Program Death-1 (PD-1) receptor, and pro- apoptotic genes, and high levels of expression of genes associated with MHC-I and II antigen. Since, protection from Simian AIDS was associated with high titers of antibodies to the SIV Envelope protein, durable effector SIV-specific CD8+ T-cells that express less pro-apoptotic genes and more genes associated with MHC-I or II antigens presentation, suggest the hypothesis that presence of antibodies may improve the quality and potency of protective SIV-specific CD8+ T-cell response.Approach 4: We have demonstrated that TRIM5alpha does not affect SIVmac251replication in vaccinated or unvaccinated Indian rhesus macaques. We determined the distribution of TRIM5alpha alleles by PCR and sequence analysis of the B30.2/SPRY domain in a cohort of eighty-two macaques. Our results demonstrated that TRIM5alpha genotype does not confound results of mucosal infection of rhesus macaques with SIVmac251.Approach 5: The ER-resident chaperone gp96, when released by cell lysis, induces an immunogenic chemokine signature and causes innate immune activation of DC and NK cells. We demonstrated that intraperitoneal immunization with a genetically engineered, secreted form of gp96, gp96-Ig chaperoning SIV antigens, induces high levels of antigen specific CD8 CTL in the rectal and vaginal mucosa of rhesus macaques. The frequency of SIV Gag- and SIV Tat-tetramer positive CD8 CTL in the intestinal mucosa reached 30-50% after the third immunization. Tetramer positive CD8 CTL expressed appropriate functional (granzyme B) and migration markers (CD103). The polyepitope specificity of the mucosal CD8 and CD4 response is evident from a strong, multifunctional cytokine response upon stimulation with peptides covering the gag, tat and env proteins. Induction of powerful mucosal effector CD8 CTL responses by cell-based gp96(SIV)-Ig immunization may provide a pathway to the development of safe and effective SIV/HIV vaccines.Approach 6: We tested the immunogenicity of a vaccine that uses human papillomavirus-based gene transfer vectors, also called pseudovirions (HPV PsVs), to deliver SIV genes to the vaginal epithelium. Our findings demonstrate that this vaccine platform induces gene expression in the genital tract in both cynomolgus and rhesus macaques. Intravaginal vaccination with HPV16, HPV45, and HPV58 PsVs delivering SIV Gag DNA, induced vaginal Gag-specific IgA, serum IgG, and T-cell responses in blood, vaginal mucosa, and draining lymph nodes that rapidly expanded following intravaginal exposure to SIVmac251. HPV PsVs-based vehicles are immunogenic, warrant further testing as vaccine candidates for HIV. Approach 7: We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV89.6P replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as two weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by four weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R = -0.83, p 0.015), and ADCVI (R = -0.84 p=0.044). These data suggest that anti-envelope antibodies with neutralizing and non-neutralizing FcgR-dependent activities may be important in the control of SHIV replication. Approach 8: The licensed smallpox vaccine, ACAM2000, is a cell culture derivative of Dryvax. Both ACAM2000 and Dryvax are administered by skin scarification and can cause progressive vaccinia, with skin lesions that disseminate to distal sites. We have investigated the immunologic basis of the containment of vaccinia in the skin with the goal to identify safer vaccines for smallpox. Macaques were depleted systemically of T- or B-cells and vaccinated with either Dryvax or an attenuated vaccinia vaccine, LC16m8. B-cell depletion did not affect the size of skin lesions induced by either vaccine. However, while depletion of both CD4+ and CD8+ T-cells had no adverse effects on LC16m8-vaccinated animals, it caused progressive vaccinia in macaques immunized with Dryvax. As both Dryvax and LC16m8 vaccines protect healthy macaques from a lethal monkeypox intravenous challenge, our data identifies LC16m8 as a safer and effective alternative to ACAM2000 and Dryvax vaccines for immune compromised individuals.